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SFM Quiz 2 > Molecular Diagnostics > Flashcards

Molecular Diagnostics Flashcards

1
Q

This type of technique used to analyze proteins allows you to look at all proteins based on their size.

A

SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis)

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2
Q

SDS is largely hydrophobic with a negative charge. It will _______ proteins and give them all a uniform ________ charge, so they will all migrate toward a ________ charge in the presence of current.

A

Unfolds
Negative
Positive

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3
Q

The next step after SDS is applied, ______-______ is used to reduce disulfide bonds between multi-subunit proteins, further denaturing it.

A

Beta-mercaptoethanol

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4
Q

This type of technique uses antibodies to detect specific proteins (done after SDS-Page has separated the proteins by their size). The proteins are transferred from gel to a nitrocellulose membrane to make them surface exposed. A primary Ab is added, then a secondary Ab that has an enzyme tag. Substrate is then added, which gives color.

A

Western blotting (aka Immunoblotting)

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5
Q

Western blotting will use a ________ antibody that binds to a specific antigen (protein), then a ________ antibody is used to bind to the _______ antibody.

A

Primary
Secondary
Primary

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6
Q

The secondary antibody is important because it is covalently coupled to a ________ molecule that makes it readily detectable. Commonly used are fluorescent dye, or the enzyme horseradish peroxidase.

A

Marker

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7
Q

This is an immunological technique that tests for the levels of a specific antigen OR antibody concentrations in biological samples using a corresponding antibody or antigen.

A

ELISA (Enzyme-linked Immunosorbent Assay)

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8
Q

In ELISA, if the antibody or antigen is present within a sample, the sample will do what?

A

Change color (due to reactive enzyme)

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9
Q

This type of ELISA measures the amount of an antibody in a sample.

A

Indirect ELISA

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10
Q

This type of ELISA measures the amount of an antigen in a sample.

A

Sandwich ELISA

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11
Q

What is an example of a Sandwich ELISA reaction?

A

Pregnancy test

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12
Q

In a pregnancy test, the reaction site (where you pee) contains free _______ antibodies which bind to the _______ present in urine.

A

hCG

hCG

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13
Q

In a pregnancy test, at the _______ site there is immobilized hCG antibodies. The hCG/antibody complex from the previous reaction site will then bind to the immobilized hCG antibody, completing the sandwich. (Line shows up or not)

A

Test

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14
Q

In a pregnancy test, at the ________ site a non-specific antibody is immobilized and dye gives color regardless of +/- hCG. This confirms the test is working.

A

Control

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15
Q

This technique is used to amplify (multiply) isolated DNA regions.

A

PCR (Polymerase Chain Reaction)

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16
Q

What are the 3 steps of PCR?

A

1) Heat to separate DNA strands
2) Cool to anneal forward and reverse primers
3) DNA synthesis (Add DNA polymerase and dNTPs)

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17
Q

PCR reactions are carried out in what?

A

Thermocycler

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18
Q

What are the advantages of PCR?

A

1) Very small start template of DNA needed

2) Large amplification from trace amount of DNA

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19
Q

What are the disadvantages of PCR?

A

1) Need to know sequence of flanking DNA for primers
2) Error prone
3) Amplification of contaminating DNA

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20
Q

HIV can be confirmed after performing Indirect ELISA by the use of _______ _______. Circulating levels of HIV -24 surface antigen appear even before Ab are formed.

A

Western blotting

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21
Q

__________ PCR (qPCR) is used to quantify the copy number of a specific gene in two or more samples in real time. In addition to primers, this technique includes a probe which fluoresces only in the presence of the PCR product. Probe is usually a complementary fragment with a fluorescent tag.

A

Quantitative

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22
Q

qPCR is used to detect levels of an ________ agent or determine levels of gene expression.

A

Infectious

23
Q

This technique can be used in forensic analysis, paternity testing, and disease detection. It detects difference in patterns of DNA within recognition sequences for restriction enzymes. (DNA finger printing)

A

Restriction Fragment Length Polymorphism (RFLP)

24
Q

For example, RFLP is used to detect presence of _______ _______ disease in a daughter from a mother without it and father with the disease. Normal beta-globulin allele has 3 Ddel restriction sites, and patients with this disease only have 2 restriction sites. Cleavage of genomic DNA followed by Ddel. Followed by electrophoresis and Southern blotting including a B-globulin-specific probe.

A

Sickle cell

25
Q

When genomes form 2 individuals are cleaved with a set of __________ enzymes and the fragments resolved on a gel, the pattern is different. That’s how forensic scientists can match an evidence sample with a suspect sample, because they will match if that person did it (hence name DNA finger printing).

A

Restriction

26
Q

These 2 techniques can be used in identification and severity of inherited diseases (Huntington disease, Fragile X syndrome, Frederich Ataxia, etc.) They look at the number of repeats of certain nucleotides within restriction sites.

A

Variable number of tandem repeats (VNTR)

Short tandem repeats (STR)

27
Q

These are state of the art biotechniques used to detect infectious agent and diagnose inherited disorders.

A

Hybridization

Polymerase Chain Reaction (PCR)

28
Q

This technique is performed with single stranded DNA binding to another strand of DNA or RNA with complementary sequence to form DNA-DNA hybrid or DNA-RNA hybrid. Useful for detection and quantification of target DNA or RNA in sample containing a mixture of other DNA/RNA.

A

Hybridization (Southern and Northern Blotting)

29
Q

In this type of hybridization, both probe and target nucleic acid are DNA.

A

Southern Blotting

***Remember SNOW DROP

30
Q

In this type of hybridization, the probe is single stranded DNA and target is mRNA.

A

Northern Blotting

31
Q

Describe the process of hybridization (southern and northern blotting).

A

First, a sample of DNA is cut into fragments then put into gel electrophoresis. The DNA is separated based on size, into single stranded fragments. These fragments are then transferred to a membrane (nylon or nitrocellulose) aka blotting. Complementary probes of DNA are then added to try and match with the particular gene of interest. Solid lines will show up on the membrane where the probe reacts with the target DNA fragment. Radioactivity from probes that react with target DNA are the only visible bands. Gene is identified. (This is southern blotting, for northern it is the same process but the target is RNA)

32
Q

This type of blotting measures the amount of protein or antibody present.

A

Western blotting

33
Q

This type of blotting detects post-translational modifications (PTMs) on proteins (i.e., lipid, carb, phosphorylation)

A

Eastern blotting

34
Q

This is the type of polymerase used in PCR to synthesize copies of DNA by extending the primers on both ends.

A

Taq polymerase

35
Q

What is the purpose of using cell-free cloning with PCR?

A

– Earlier detection of microorganisms (HIV, bacterial and fungal infections that cause Lyme disease)

– Detection of specific genetic mutations (Cystic fibrosis, Thalassemia, Hereditary hemochromatosis, Huntington’s, Fragile X syndrome)

36
Q

T/F. qPCR takes “snapshots” of levels in real-time via the level of fluorescence present. Fluorescence is more intense as double-stranded DNA is made.

A

True

37
Q

On a qPCR graph of a cancer cell sample vs. normal cell sample, an arbitrary threshold is reached in 20 cycles by cancers cells compared to 28 cycles in control cells. This means cancer cells have higher amounts of what?

A

Target DNA

38
Q

To detect variations in DNA sequences, these tests are used. Their uses are forensics and diagnostics for prenatal diagnosis (amniocentesis), newborn screening (IEM), and genetic carriers.

A

RFLP (Restriction fragment length polymorphism)

VNTR (Variable number of tandem repeats)

39
Q

How is cDNA made?

A

Take amino acid sequence from protein and find the mRNA, then use reverse transcriptase to develop a complementary single-stranded DNA sequence from the mRNA (aka cDNA). DNA polymerase then creates double-stranded DNA from the cDNA. This DNA is inserted into a vector, where high levels of replication can occur. This cDNA sequence can be put into a “DNA library” so we know sequences for certain proteins, etc.

40
Q

Large scale production of ________ proteins can produce insulin, growth hormone, erythropoietin, clotting factors, and vaccines against diseases (like flu, malaria, viral infections).

A

Recombinant

41
Q

For recombinant proteins, _______ of the proteins is inserted into expression vectors. These are engineered to allow high levels of replication, transcription and translation to occur in a host. Large scale production and purification occurs.

A

cDNA

42
Q

Put the following steps of production of insulin in order:

A. A plasmid DNA is extracted from a bacterium and cut with restriction enzyme, forming plasmid vector.

B. Recombinant DNA introduced into a bacterial cell to form the recombinant bacterium.

C. The recombinant bacteria multiply in a fermentation tank and price human insulin.

D. Insulin-producing gene is isolated from pancreas cells.

E. Human insulin-producing gene is inserted into the bacterial plasmid vector to form the recombinant DNA.

F. Insulin is extracted, purified and packaged for use by diabetic patients.

A

1) D.
2) A.
3) E.
4) B.
5) C.
6) F.

43
Q

To improve on insulin, we need to make modified forms with better biological activity. Normal human insulin has _______ at position 28 and _______ at position 29 at C terminus of B chain. _______ (Eli Lilly) has reverse position of these amino acids, and _______ _______ has proline 28 replaced by aspartic acid.

A

Proline
Lysine
Lispro
Insulin aspart

44
Q

Lispro and Insulin aspart are faster acting, and more readily absorbed insulins. If these are mixed with normal insulin then it provides a longer range of ________ control.

A

Glycemic

45
Q

For flu vaccine production, the FDA distributes samples, or seed virus, of the strains to the manufacturers. Seed virus is injected into _______ _______. Each virus strain is made separately and later combined to make one vaccine.

A

Chicken eggs

***Virus strains multiply in incubated eggs, then strains are later blended together to form one vaccine

46
Q

The production of _________ are used as drugs (for cancer, rheumatoid arthritis, Crohn’s disease, hepatitis) and for research purposes.

A

Antibodies

47
Q

This type of antibody is specific for a single epitope on antigen.

A

Monoclonal Ab

48
Q

Single clones of B lymphocytes are fused with a tumor cell to make it…

A

Immortalized

49
Q

Put the following steps of antibody production in order:

A. Most efficient hybridoma clones producing most effective antibodies are chosen. These are grown in culture and antibody harvested.

B. Hybridomas screened for antibody production

C. Hybridoma secreting antibody produced

D. Ab secreting cells fused with immortalized tumor cells via PEG detergent

E. Immunization of mouse with immunogen peptide/protein

F. Antibody producing hybridoma cloned (One cell clone will secrete one type of antibody)

G. Ab secreting cells harvested from Spleen (B cells, Plasma cells)

A

1) E.
2) G.
3) D.
4) C.
5) B.
6) F.
7) A.

50
Q

Put the following steps of ELISA in order:

A. Unbound/nonspecifically bound molecules are washed away.

B. A second antibody conjugated with horseradish peroxidase (HRP) is added and binds to the captured analyte.

C. Blue color is developed proportionally to the amount of analyte in the sample.

D. After the unbound second antibody is washed away (HRP substrate), tetramethylbenzidine (TMB) is added to the wells.

E. Any analyte (i.e., target protein) from standard or biological samples will be captured (bound) to the antibody when added to the well.

F. A microplate is pre-coated with a capture antibody, which is immobilized to the plate’s surface.

A

1) F.
2) E.
3) A.
4) B.
5) D.
6) C.

51
Q

ELISA can be used to diagnose ______. Specific antibodies for this are produced in humans 4-6 weeks after infection. ELISAs are used for detection due to their efficacy, sensitivity, and cost effectiveness (can produce false-positives and false-negatives, need confirmation with western blotting).

A

HIV

***specifically Indirect ELISA (detects antibodies)

52
Q

ELISA can be used for MI detection as well. _______ is a protein present in striated muscle such as heart, and is attached to ________.

A

Troponin

Tropomyosin

53
Q

In ELISA MI detection, the troponin has 3 subunits, ____, ____, and ____. Cardiac forms of _____ and _____ increase in acute MI. This is measured by ELISA. Serves as useful markers of ischemia along with other protein markers.

A

I; T; C

I; T

54
Q

ELISA used to measure cardiac Troponin T is done by taking a biological sample from patient at various times before and after an acute MI. Immobilized antibodies will recognize the human cardiac Troponin T. This is (INDIRECT/SANDWICH) ELISA.

A

Sandwich

SFM Quiz 2 (12 decks)

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